RESEARCH ARTICLE 2721
Development 139, 2721-2729 (2012) doi:10.1242/dev.077206
© 2012. Published by The Company of Biologists Ltd
Fat facets deubiquitylation of Medea/Smad4 modulates
interpretation of a Dpp morphogen gradient
Michael J. Stinchfield1, Norma T. Takaesu1, Janine C. Quijano1, Ashley M. Castillo1, Nina Tiusanen2,
Osamu Shimmi2, Elena Enzo3, Sirio Dupont3, Stefano Piccolo3 and Stuart J. Newfeld1,*
SUMMARY
The ability of secreted Transforming Growth Factor  (TGF) proteins to act as morphogens dictates that their influence be strictly
regulated. Here, we report that maternally contributed fat facets (faf; a homolog of USP9X/FAM) is essential for proper
interpretation of the zygotic Decapentaplegic (Dpp) morphogen gradient that patterns the embryonic dorsal-ventral axis. The data
suggest that the loss of faf reduces the activity of Medea (a homolog of Smad4) below the minimum necessary for adequate Dpp
signaling and that this is likely due to excessive ubiquitylation on a specific lysine. This study supports the hypothesis that the control
of cellular responsiveness to TGF signals at the level of Smad4 ubiquitylation is a conserved mechanism required for proper
implementation of a morphogen gradient.
KEY WORDS: USP9X/FAM, Smad4/Medea, TGF/BMP, Deubiquitylation, Dorsal-ventral axis, Drosophila
mesoderm induction (Dupont et al., 2005; Dupont et al., 2009). In
mouse embryos, epiblast-specific Ecto (Trim33 – Mouse Genome
Informatics) knockout leads to upregulation of Nodal-dependent
mesoderm-induction (Morsut et al., 2010). At present it is unclear
whether this mechanism for Smad4 regulation is truly general or
vertebrate specific.
The Smad4 deubiquitylase USP9X/FAM is homologous to
Drosophila fat-facets (faf), suggesting conservation of Smad4/Med
regulation by deubiquitylation (supplementary material Fig. S1)
(Chen et al., 2000). This hypothesis is supported by studies in
Drosophila where expression of Xenopus Ecto led to phenotypes
similar to mutations in Med that were rescued by co-expression of
Drosophila Faf (Dupont et al., 2009). In addition, sequence
analysis (Konikoff et al., 2008) found that a conserved lysine
(Lys519 in human Smad4; Lys738 in Med) is the residue through
which Ecto and USP9X regulate Smad4 activity (Dupont et al.,
2009). To date, no developmental roles for Faf in TGF signaling
have been identified via mutational analyses in any species.
Here, we report the maternal and zygotic requirement for faf in
Dpp signaling during DV patterning. We analyzed faf
transheterozygous genotypes that generate embryos capable of
surviving beyond the faf-null phenotype of blastoderm arrest. We
found that a subset of these are dominant maternal enhancers of
dpp mutations that engender defects in DV axis formation. We
were able to rescue faf enhancement of dpp with a nonubiquitylatable form of Med. Taken together, the data suggest an
important developmental role for Faf as a Med deubiquitylase
during Dpp DV signaling. Overall, our study reveals that
deubiquitylation is a highly conserved mechanism employed by
cells to fine-tune their interpretation of TGF signals.
MATERIALS AND METHODS
Fly stocks
1
School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA.
2
Institute of Biotechnology, University of Helsinki, Helsinki, Finland. 3Department of
Biomedical Sciences, University of Padova Medical School, Padova, Italy.
*Author for correspondence ([email protected])
Accepted 10 May 2012
Mutant strains are: fafBP, fafF08, fafB3, fafBX3, fafB4, fafB5, fafB6 (Fischer-Vize
et al., 1992), fafEP381 (Berger et al., 2001), dpphr4, dpphr27, dppHin61 (St
Johnston et al., 1990), sogy506 (Ferguson and Anderson, 1992), Med8
(Wisotzkey et al., 1998), Med15, Med17 (Hudson et al., 1998) and Mad12
(e.g. Sekelsky et al., 1995). Transgenic strains are: nos.Gal4:VP16-MVD1
(van Doren et al., 1998), UASp.GFP-Tub84B (Grieder et al., 2000) and
act-lacZ-CB1 (Bourgouin et al., 1992).
DEVELOPMENT
INTRODUCTION
TGF signals are pleiotropic regulators of animal development. In
Drosophila, the TGF family member Dpp, a homolog of
vertebrate BMP2/4, initiates signal transduction with a complex of
receptor kinases. One of the receptors then phosphorylates Mad, a
member of the Smad family of TGF signal transducers. Once
phosphorylated, Mad translocates to the nucleus, forms a complex
with its sister Smad protein Medea (Med) and regulates gene
expression in cooperation with tissue-specific co-factors (Derynck
and Miyazono, 2008).
During early embryogenesis, Dpp plays a key role in
orchestrating dorsal-ventral (DV) axis formation. Prior to dpp
zygotic transcription, maternally contributed Mad and Med are
present in every cell. At cellular blastoderm stage, a dorsal to
ventral extracellular gradient of Dpp protein is generated via a
system of extracellular regulation that is translated quantitatively
by each cell into intracellular levels of phosphorylated Mad
(pMad). At specific thresholds of Mad phosphorylation, cells
implement distinct developmental programs. Perturbation of the
Dpp gradient (dpp mutations) or its interpretation (Mad or Med
mutations) results in aberrant cell fate decisions and abnormal
development (Shimmi et al., 2005).
Recent evidence indicates that Med (a homolog of vertebrate
Smad4) is also a tunable determinant of cellular responses to TGF
signals. In vertebrate embryos, Smad4 monoubiquitylation opposes
the formation of Smad complexes, providing a mechanism by
which nuclei monitor the presence of extracellular ligands and
activate gene expression appropriately. In Xenopus, knockdown of
the Smad4 ubiquitin ligase Ectodermin (Ecto/TRIM33/Tif1-)
causes expansion of mesoderm markers, whereas knockdown of
the Smad4 deubiquitylase USP9X/FAM causes defective
Genetics
Assays of dominant maternal enhancement, zygotic lethality, maternal
effect lethality, synthetic lethality and transgenic rescue of faf enhancement
were conducted using standard methods (Sekelsky et al., 1995). Stage of
lethality tests were as described (Takaesu et al., 2006). Alignments and
phylogenetic trees were as described previously (Konikoff et al., 2010).
Cuticles and embryos
Cuticle scoring followed Wharton et al. (Wharton et al., 1993). Nonfluorescent antibody labeling followed Johnson et al. (Johnson et al., 2007).
Double labeling employed anti-lacZ (Organon Teknika) and anti-Hnt
(DSHB- 1G9) detected with biotinylated goat anti-mouse or anti-rabbit and
Vecta Stain Elite (Vector Labs). RNA in situ double labeling of embryos
with rho or dpp or sog and lacZ cDNAs or antibody double labeling with
anti-pMad and anti-lacZ were as described previously (Takaesu et al.,
2002; Shimmi et al., 2005). Fluorescence double labeling of embryos
followed methods of Quijano et al. (Quijano et al., 2010) using anti-Hnt
and anti-lacZ. Additional antibodies employed in unfertilized eggs and
embryos were anti-Flag (Sigma) and anti-Bonus (Beckstead et al., 2001).
Secondary antibodies were Alexa Fluor goat anti-rabbit, anti-mouse and
anti-guinea pig (Molecular Probes).
Transgenes
A Med-K738R cDNA was generated by site directed mutagenesis from
Med-wt in pAcpA (McCabe et al., 2004). These were subcloned as MluINheI fragments into a modified pUASP vector (Rørth, 1998) with a novel
multiple cloning site. Human Smad4-K519R and Smad4-wt cDNAs in
pRK5 (Dupont et al., 2009) were excised with EcoRI-SalI and subcloned
as EcoRI-PmeI fragments into the modified pUASP vector. All UASP
plasmids were verified by sequencing prior to generating multiple
transgenic lines by standard methods.
RESULTS
Given that USP9X deubiquitylates Smad4, we tested the
hypothesis that faf, the fly USP9X homolog, plays a role in Dpp
signal transduction, a process in which Med is a major component.
Two events that depend on Dpp signaling are adult wing vein
formation and DV patterning in the early embryo. A study of wings
from adults generated by a set of inter se crosses between eight faf
alleles did not identify any defects. We did note that
transheterozygous faf mutant females are sterile, as reported by
Fischer-Vize et al. (Fischer-Vize et al., 1992). This prompted us to
examine the possibility that faf plays a role in Dpp signaling during
embryonic DV patterning.
faf mutations are dominant maternal enhancers
of dpp mutations
We began with the classic assay for dominant maternal
enhancement of dpp recessive lethal mutations that led to the
discovery of Mad and Med (Sekelsky et al., 1995; Raftery et al.,
1995). Missense mutations in the Dpp pro-domain (dpphr4; G402E)
(Wharton et al., 1996) impact ligand cleavage, dimerization or
stability and are almost completely recessive; they survive at near
wild-type levels when heterozygous but are absolutely lethal when
homozygous. In this assay, a female parent with a heterozygous
mutation at a second locus reduces the survival of the dpp recessive
allele as a heterozygote from near wild type to near absolute
lethality. For Mad and Med, the explanation is that the reduction in
the dose of functional maternal RNA for either of these signal
transducers in combination with a reduction in the dose of fully
functional Dpp leads to a diminished Dpp morphogen gradient,
ventralization of the embryo and death.
Employing females heterozygous for seven of our eight faf
alleles (fafEP381 is a P insertion in the first intron creating a viable
allele that was tested as a homozygote), we found that fafEP381
Development 139 (15)
displayed maternal enhancement of dpphr4 and that fafF08 (missense
mutation in the catalytic histidine) displayed dominant maternal
enhancement. Mating to fafEP381 homozygous females reduced the
survival of dpphr4 progeny to 26% of expected. For comparison,
mating to Med15 and Med17 (missense mutations) homozygous
females reduced the survival of dpphr4 to less than 5% of expected.
Mating to fafF08 heterozygous females reduced dpphr4 survival to
10% of expected. For comparison, mating to Med8 (nonsense
mutation) heterozygous females reduced dpphr4 survival to 5% of
expected (Fig. 1E; supplementary material Table S1A). Stage of
lethality assays revealed that fafF08 enhancement of dpphr4 led these
individuals to die as embryos and that there was no effect on dpphr4
survival in the reciprocal cross between fafEP381 males and dpphr4
females (supplementary material Table S2). These results are
consistent with data for both Mad and Med (Sekelsky et al., 1995;
Raftery et al., 1995).
We then analyzed cuticles from the two faf maternal
enhancement crosses and compared them with dpphr4 homozygous
and Med enhanced dpphr4 cuticles. The analysis revealed that faf
enhanced dpphr4 progeny displayed defects in DV patterning
similar to those of ventralized dpphr4 homozygous and Med
enhanced dpphr4 progeny (Fig. 1A-D; supplementary material Table
S3A). Shared defects include a herniated head skeleton at the
anterior, misshapen and/or internalized Filzkorper at the posterior,
dorsal extension of the ventral denticle belts and a partially Ushaped body. These similarity of faf enhanced dpphr4 embryos to
dpphr4 homozygotes and Med enhanced dpphr4 embryos suggest
that maternal Faf deubiquitylation is important for Dpp signaling
during DV patterning.
faf zygotic mutants display DV defects
The maternal enhancement experiments showed that faf mutations
can sensitize embryonic development to defective Dpp signaling.
Therefore, we sought to determine whether faf mutations alone can
cause defects comparable with mutations in the Dpp pathway. In
these studies, we analyzed both transheterozygous zygotic and
maternal mutants by assessing survival and cuticle phenotypes. We
employed an inter se strategy with eight faf mutant alleles to generate
faf transheterozygous zygotic mutant progeny (e.g. fafF08/fafB3). We
found that many faf zygotic mutant combinations are fully viable but
others generate adults at less than 50% of the expected ratio. In three
genotypes, only 25% of the expected faf mutants survived to
adulthood (Fig. 1J; supplementary material Table S1B). A reduction
in faf zygotic mutant survival had not been noted before.
In cuticle studies of genotypes with reduced survival, we
observed that faf transheterozygous zygotic mutants displayed a
range of defects in DV patterning (Fig. 1F-I; supplementary
material Table S3B). The most severely affected faf zygotic
mutants (fafF08/fafB3, 27% of expected) displayed DV defects that
resembled dpp null genotypes. Other faf zygotic mutants generated
cuticles similar to dpphr4 homozygous and dpphr4 enhanced
cuticles. This data suggests that, in addition to the maternal
requirement identified in enhancement assays, there is also a
zygotic requirement for faf in Dpp signaling during DV patterning.
faf maternal mutants form an allelic series that
generates a gradient of DV defects
We continued the inter se experiment for a second generation to
confirm a maternal requirement for faf in Dpp DV signaling. We
employed transheterozygous females bearing all combinations of
the eight faf mutants in crosses to wild-type males. The progeny
will be heterozygous for a faf mutant allele derived from their
DEVELOPMENT
2722 RESEARCH ARTICLE
Faf deubiquitylation in Dpp signaling
RESEARCH ARTICLE 2723
Fig. 1. faf enhancement of dpp and faf zygotic lethality is
associated with DV defects. Cuticles in lateral view with anterior
leftwards and dorsal upwards. The maternally contributed allele is listed
first. (A)Wild type. Broad white ventral denticle belts (bottom), narrow
white Filzkorper (upper right corner) and internal head skeleton (left
side) are visible. (B)Homozygous dpphr4 ventralized cuticle with a short
curved body, dorsally extended denticles, herniated head and defective
Filzkorper. (C)Dominant maternal enhancement of dpphr4 by Med8 is
visible in a ventralized Med8 and dpphr4 double heterozygous cuticle
similar to dpphr4. (D)Maternal enhancement of dpphr4 by fafEP381 is
visible in a ventralized fafEP381 and dpphr4 double heterozygous cuticle
similar to the dpphr4 and the Med8 and dpphr4 double heterozygous
cuticle. (E)Maternal enhancement of dpphr4 by Med and faf females.
The percentage of expected dpphr4 progeny from crosses to Med or faf
heterozygous females is compared with mating with wild-type females
(first bar). Numerical data are in supplementary material Table S1A.
(F)dppHin61 homozygous null cuticle is fully ventralized with a herniated
head, ectopic denticle belt replacing the Filzkorper and ventral denticle
belts encircling the body. (G)fafF08/fafB3 ventralized cuticle with dorsally
extended denticles and an ectopic denticle belt replacing the Filzkorper.
(H)fafF08/fafBP ventralized cuticle with dorsally extended denticles and
poorly developed Filzkorper. (I)fafBP/fafB3 ventralized cuticle with
herniated head, dorsally extended denticles and misplaced Filzkorper.
(J)Zygotic lethality of faf transheterozygous genotypes. The percentage
of expected faf progeny from representative crosses is compared with
those generated by a faf null allele mated to wild type (first bar).
Numerical data are in supplementary material Table S1B.
mother and a wild-type faf allele from their father. The paternal
wild-type allele allows normal development and defects are
attributed to the faf mutation in the maternal parent.
Embryos derived from faf homozygous mutant females
primarily die prior to the onset of embryonic dpp expression
(Fischer-Vize et al., 1992). However, our second-generation crosses
faf mutations enhance Med mutations and can
partially suppress sog mutations
If the mechanism by which USP9X deubiquitylates Smad4 is
operating in Dpp DV signaling, then one prediction is that reducing
faf and Med dose together in double heterozygous embryos will
reduce the amount of functional Med to the point of interfering
with Dpp signaling. A corollary is that if the effect of the double
heterozygote is non-reciprocal, then the more crucial component is
the one that must be compromised in the mother to obtain an effect.
In our analysis, we observed that a subset of faf and Med double
heterozygous genotypes displayed the synthetic lethality predicted
by the USP9X-Smad4 mechanism. In these cases, fewer than half
the expected number of double heterozygous progeny survived (Fig.
2J; supplementary material Table S1C). In all combinations with
synthetic lethality, the female parent was heterozygous for Med8
while the male parent contributed a faf mutant allele, indicating that
maternal reduction in Med is more damaging to Dpp DV signaling
than maternal reduction in faf. Cuticle studies of double heterozygous
genotypes with reduced survival revealed that the lethality was due
to defects in DV patterning (Fig. 2H,J; supplementary material Table
S3C). Data prior to this point revealed that faf plays a role in Dpp
DV signaling and now faf-Med synthetic lethality suggests a
mechanism: faf impacts Dpp DV signaling via interactions with Med.
This suggestion is supported by results from crosses of faf mutants
and Mad12 (null allele for Dpp signaling) (Sekelsky et al., 1995). All
Mad and faf double heterozygous genotypes displayed normal
survival and wild-type cuticles.
We then examined the ability of faf to suppress dorsalization
defects caused by to mutations in sog. If dpp mutations are able to
partially suppress sog mutant phenotypes (Francois et al., 1994),
then mutations affecting Dpp DV signaling pathway components
will have the same effect. We validated the prediction with a
mutation in Med and found that strong faf mutant alleles have the
same effect (Fig. 2K-L; supplementary material Table S3D). Taken
together, our survival assays and cuticle data suggest that maternal
and zygotic faf activity plays a positive role in Dpp DV signaling
via interactions with Med.
Loss of hindsight and rhomboid in faf mutants
phenocopy Dpp signaling mutants
To confirm the role of faf in Dpp signaling, we next visualized
established Dpp target genes (Hindsight and rhomboid) in genetic
combinations displaying cuticular DV defects. Hin is expressed in
DEVELOPMENT
revealed that embryos from a subset of faf transheterozygous
mutant females survive past this point and that their cuticles display
DV defects similar to ventralized dpp mutants. Importantly, cuticles
derived from embryos generated by these faf mutant females could
be ordered into an allelic series. As opposed to the zygotic
requirement for faf in DV patterning, which was revealed by
combinations of strong hypomorphic alleles, the graded maternal
requirement for faf was identified in combinations of weak
hypomorphic alleles (Fig. 2A-G).
The least affected cuticles were derived from fafB4/fafB5 females.
Next in severity are fafB4/fafB6, fafF08/fafB4 and fafB4/fafBX3 cuticles.
The most severe DV defects are seen in fafB3/fafB5 cuticles.
Embryos derived from stronger transheterozygous combinations,
such fafB6/fafBP, do not generate any cuticle, owing to early
embryonic lethality associated with the faf-null phenotype of
blastoderm arrest. The faf maternal mutant allelic series creates a
gradient of DV defects that parallels the phenotypes of an allelic
series constructed for dpp (Wharton et al., 1993).
the amnioserosa, the dorsal-most embryonic tissue and the one that
requires the highest level of Dpp signaling (e.g. Raftery et al.,
1995). Each of the Hin experiments was consistent with cuticle
data derived from the same cross (Fig. 3A-G).
The dominant maternal enhancement of dpphr4 by fafF08 led to a
loss of Hin-expressing cells, though less severe in this example
than Med17 enhancement of dpphr4 and homozygosity for dpphr4.
Zygotic transheterozygous combinations of strong faf mutant
alleles also displayed reduced Hin expression. Embryos derived
from faf maternal transheterozygous mutants for which a subset
survive past the null phenotype exhibit essentially no Hin
expression. This closely resembles dpphr4 homozygous embryos
and Med mutant germline clones (Hudson et al., 1998). Med
maternal and faf paternal double heterozygous embryos display
reduced Hin expression whereas embryos from the reverse cross
(Med paternal and faf maternal) are wild type.
We extended the analysis to rhomboid (rho) expression in the
dorsal ectoderm of cellular blastoderm (stage 5) embryos: rho is
the earliest known target of Dpp signaling in embryonic DV
Development 139 (15)
Fig. 2. faf maternal mutants generate a gradient of DV defects,
whereas faf zygotic heterozygosity partially enhances Med and
suppresses sog mutants. Cuticles in lateral view derived from mating
of the indicated faf transheterozygous female to a wild-type male.
(A)Wild type. (B)fafB4/fafB5 cuticle displays modest ventralization with a
herniated head, poorly developed Filzkorper and slightly extended
ventral cuticles similar to a haploinsufficient dppHin46 heterozygous
cuticle (Wharton et al., 1993). fafB4 is an in-frame six-residue insertion
at position 279 and fafB5 is a frame-shift after amino acid 2150. These
widely spaced alternations each generate a weak hypomorphic allele
(Fisher-Vize et al., 1992; Chen and Fisher, 2000). (C)fafB4/fafB6 cuticle
displays increased ventralization with a herniated head, defective
Filzkorper and denticle belts that extend throughout the ventral half of
the cuticle similar to a dpphr56 homozygous cuticle. fafB6 is a nonsense
mutation at position 459 that functions as an near protein-null allele.
(D)fafF08/fafB4 cuticle displays significant ventralization with a herniated
head, defective Filzkorper, malformed dorsal cuticle and denticle belts
that extend well into the dorsal half of the embryo similar to a dpphr92
homozygous cuticle and to cuticles from Med mutant germline clones
with partial paternal rescue (Hudson et al., 1998). fafF08 is a missense
mutation in a catalytic histidine (H1986Y) that generates an allele
impacting Dpp signaling, as assayed by dpphr4 enhancement.
(E)fafB4/fafBX3 cuticle also displays significant ventralization with a
herniated head, defective Filzkorper, malformed dorsal cuticle and
extended denticle belts similar to a dppHin46/dppe87 cuticle. fafBX3 is an
in-frame deletion of 15 bp shortly after the catalytic domain that
generates a strong hypomorphic allele. (F)fafB3/fafB5 cuticle appears
almost fully ventralized within the vitelline membrane. Its rudimentary
cuticle contains one anterior denticle belt that fully encircles the
embryo (indicated by the white arrowhead on the left outside the
vitelline membrane) similar to a dppHin94/dppHin95 cuticle. fafB3 is a
nonsense mutation at position 71 that generates a protein null allele.
(G)fafB6/fafBP embryo remains intact inside the vitelline membrane but
contains no cuticle due to early embryonic lethality associated with the
faf-null phenotype of blastoderm arrest. (H)faf mutation contributed
by a heterozygous mother yields a fafB4 and Med8 double heterozygous
cuticle that appears wild type. Note that Med and faf are both on
chromosome 3, so we do not employ ‘+’ to represent the homolog,
even though Med on the fafB4 chromosome is normal, as is faf on the
Med8 chromosome. (I)Med mutation contributed by a heterozygous
mother yields a Med8 and fafB4 double heterozygous cuticle that is
similar to dpphr4. (J)Maternal enhancement of faf mutants by Med8
heterozygous females. The percentage of expected Med8 and faf
mutant double heterozygous progeny is compared with those
generated by mating of Med8 heterozygous females with wild-type
males (first bar). Numerical data are in supplementary material Table
S1C. (K)sogy506 hemizygous dorsalized cuticle containing extremely
truncated denticles, completely U-shaped body, herniated head and
Filzkorper defects. (L)sogy506 hemizygous and dpphr4 heterozygous
cuticle with partially restored denticles and curved body. (M)sogy506
hemizygous and Med15 heterozygous cuticle with partially restored
denticles, normal body shape and misplaced, misshapen Filzkorper.
(N)sogy506 hemizygous and fafF08 heterozygous cuticle with partially
restored denticles, normal body shape and poorly developed Filzkorper.
patterning (Yu et al., 2000) and assay of rho transcription is also a
standard means of evaluating Dpp activity (e.g. Ross et al., 2001).
If faf mutations influence dorsal ectoderm rho expression, this
would further support a role in Dpp DV signaling (Fig. 3H-N).
The dominant maternal enhancement of dpphr4 by fafF08 led to loss
of rho expression in the central region of the dorsal ectoderm. This
phenotype mimicked Med17 enhancement of dpphr4 and
DEVELOPMENT
2724 RESEARCH ARTICLE
Faf deubiquitylation in Dpp signaling
RESEARCH ARTICLE 2725
Fig. 3. Loss of Hnt and rho in faf mutant genotypes suggests
reduced Med function in Dpp DV signaling. (A-G)Stage 10/11
embryos in lateral view depicting Dpp-dependent Hnt expression in the
large cells of the amnioserosa. Dpp-independent Hnt expression in the
foregut and hindgut (below the plane of focus) act as an internal
control for staining. (A)Wild-type embryo. (B)dpphr4 homozygous
ventralized embryo with a few scattered amnioserosa cells. (C)Med17
and dpphr4 maternally enhanced embryo appears similar to dpphr4.
(D)fafF08 and dpphr4 maternally enhanced embryo is similar to the
ventralized Med17 and dpphr4 embryo. (E)fafBP/fafB3 embryo appears
weakly ventralized with a reduced number of amnioserosa cells.
(F)Embryo from a fafF08/fafB4 female mated to a wild-type male is
similar to dpphr4. (G)Med8 maternal and fafB4 paternal double
heterozygous embryo is similar to the weakly ventralized fafBP/fafB3
zygotic embryo but with fewer amnioserosa cells. (H-N)Stage 5
embryos in dorsal/lateral views revealing Dpp-dependent rho
transcription in the dorsal ectoderm. (H)Wild-type embryo with a
central dorsal stripe of rho expression bounded by wider domains
(arrowheads). (I)dpphr4 homozygous embryo with no central stripe of
rho. (J)Med17 and dpphr4 maternally enhanced embryo is similar to
dpphr4. (K)fafF08 and dpphr4 maternally enhanced embryo is similar to
dpphr4. (L)fafBP/fafB3 embryo appears weakly ventralized with faint rho
expression in the dorsal ectoderm. (M)Embryo from fafF08/fafB4 female
mated to a wild-type male has passed syncytial blastoderm arrest but
displays no rho expression. (N)Med8 maternal and fafB4 paternal
double heterozygous embryo is similar to the weakly ventralized
fafBP/fafB3 zygotic embryo but with further reduced rho expression.
faf mutations do not affect dpp or sog
transcription or pMad activation/dorsal
localization
We then analyzed an alternative hypothesis for the role of faf in
Dpp DV signaling. During DV patterning the roles of opposing
morphogen gradients composed of Dpp and Sog are well known.
dpp is transcribed in the dorsal half of the embryo and then posttranslational mechanisms, such as extracellular interactions with
Sog transcribed in the ventral-lateral region of the embryo, create
a gradient of Dpp activity that induces five distinct cell fates along
the DV axis (Shimmi et al., 2005). Assays of cuticles or Hin or rho
expression in faf mutants cannot formally exclude the possibility
that faf modulates DV patterning by regulating dpp and/or sog
transcription. We conducted dpp and sog situ hybridization studies
using faf maternal mutants for which a subset of embryos survive
past the null phenotype and zygotic mutants that display DV
defects. Both fafB4/fafB5 maternal and fafF08/fafBP zygotic mutant
embryos contain wild-type dpp RNA (Fig. 4A-C) as well as wildtype sog RNA (Fig. 4D-F) expression. This suggests that the
alternative hypothesis of transcriptional activation is false.
We also analyzed pMad expression in faf maternal and zygotic
mutants. This assay can provide evidence that will allow us to further
eliminate the possibility that faf influences Dpp DV signaling via
interactions with Mad, rather than Med. Both maternal and zygotic
faf mutant combinations display normal pMad activation and
localization to the dorsal-most region at stage 5 (Fig. 4G-I). These
pMad results are reproducible in multiple maternal and zygotic faf
mutants (supplementary material Fig. S2) and are similar to pMad
expression in dpphr4 heterozygous embryos that have wild-type DV
patterning (supplementary material Fig. S3). These data confirm our
hypothesis that faf blocks Dpp DV signaling downstream of Mad.
We noted that faf maternal mutants show a slightly broader pMad
stripe and believe this is due to reduced Med activity in a Dpp
feedback loop (Wang and Ferguson, 2005).
Taken together, all of the data are consistent with our hypothesis
for the role of faf in Dpp signaling: loss of maternal or zygotic faf
leads to a reduction in Med activity, insufficient Dpp signal
transduction and DV defects. Thus, Faf deubiquitylation of Med
during Dpp DV signaling is the first genetically defined
DEVELOPMENT
homozygosity for dpphr4. Zygotic transheterozygous combinations
of strong faf mutant alleles also displayed reduced rho central region
expression though less severe in this example than Med17
enhancement of dpphr4 and homozygosity for dpphr4. Embryos
derived from faf maternal transheterozygous mutants for which a
subset survive past the null phenotype exhibit no rho expression in
any region of the dorsal ectoderm, a phenotype also seen in embryos
derived from homozygous Med15 females. Med maternal and faf
paternal double heterozygous embryos display reduced rho
expression in the central region. Each of the rho experiments was
consistent with the cuticle and Hin data derived from the same cross.
We replicated all of these results with a second missense
mutation in the prodomain-dpphr27 (E316K) (Wharton et al., 1996).
For example, Med8 dominant maternal enhancement of dpphr27 led
to the survival of 3% of dpphr27 individuals, whereas fafF08
enhancement led to survival of 4%. One hypothesis that explains
all of the data is that loss of the Faf deubiquitylase reduces Med
activity below the level needed for gene expression in tissues
requiring the highest amount of Dpp. This hypothesis for the
relationship between Faf and Med in Dpp signaling is analogous to
that between USP9X and Smad4 in vertebrate TGF signaling
(Dupont et al., 2009).
2726 RESEARCH ARTICLE
Development 139 (15)
developmental event employing the conserved USP9X-Smad4
regulatory mechanism. We then wondered whether the mechanism
is conserved at the level of the deubiquitylated lysine in Med.
Medea Lys738 is deubiquitylated by maternal Faf
during Dpp DV signaling
We examined whether the USP9X-Smad4 regulatory mechanism is
conserved at the level of the deubiquitylated lysine in Med using
transgenic rescue experiments that were evaluated with survival,
cuticle and Hnt data. We compared the ability of a wild-type Med
transgene (Med-wt) to rescue faf and Med dominant maternal
enhancement of dpphr4 with the rescuing ability of a ubiquitinresistant Med transgene. Our phylogenetic analysis showed that
Lys519 in human Smad4 (targeted by Ecto and USP9X) is conserved
as Lys738 in Med (Konikoff et al., 2008). For the non-ubiquitylatable
Med transgene, we assumed the homologous lysine was the Faf
target and created Med-K738R. We used arginine as the replacement
to avoid disturbing Med protein structure with a dissimilar (nonnegatively charged) amino acid. The USP9X-Smad4 model predicts
that the non-ubiquitylatable transgene (Med-K738R) will be
hyperactive in Dpp signaling and thus rescue dpphr4 embryos from
faf or Med maternal enhancement better than Med-wt.
We conducted the rescue experiment with two different mutants
(fafF08 and Med8), at two discrete levels of transgene expression and
with two distinct sets of transgenes. Basal levels of expression are
generated by the P transposase minimal promoter plus first intron and
the K10 3⬘UTR that are present in the UASP vector (Rørth, 1998).
Overexpression is driven by nos.Gal4 (Gal4:VP16-nos.3⬘UTR, line
MVD1) (van Doren et al., 1998) from a chromosome also containing
UASP.eGFP to monitor nos.Gal4 expression. Figure S4
(supplementary material) reveals that relative levels of transgene
expression in these two genotypes are essentially identical for two
different transgene insertions (Med-wt and Med-K728R) in
unfertilized eggs and stage 5 embryos, strongly suggesting that
position effects will not interfere with this assay. Last, all balancer
chromosomes were marked with transgenic lacZ to allow positive
identification of experimental embryos during the Hnt analysis.
Results from all rescue experiments were compared with the original,
non-transgenic dpphr4 enhancement cross.
Rescue assays with fafF08 were the most informative. The logic
is that loss of the deubiquitylase will be rendered less consequential
in the presence of a non-ubiquitylatable and thus hyperactive MedK738R transgene, only if Lys738 is the ubiquitylated lysine. If any
other lysine were ubiquitylated, then the loss of faf would be
compensated for by Med-K738R at the same level as Med-wt. The
most telling result was with the basal promoter (Fig. 5G;
supplementary material Table S4A). Here, expression of Med-wt
increased the survival of dpphr4 progeny 1.6-fold. By contrast,
expression of Med-K738R increased the survival of dpphr4 progeny
4.2-fold. Thus, Med-K738R performed 2.65-fold better in the
rescue of dpphr4 with basal expression. Once the transgenes were
driven at ectopic levels with nos.Gal4, then Med-wt was equal to
Med-K738R as both reached a rescue ceiling in this assay at
roughly 80% of expected.
The survival results are supported by cuticle assays (Fig. 5A-F,
left column; supplementary material Table S5A). A high percentage
of cuticles show DV defects in the fafF08 enhancement control and
in the basal rescue experiment with Med-wt. The basal experiment
with Med-K738R shows an intermediate level and the ectopic
rescue experiments show a low percentage of cuticles with DV
defects. The cuticle data for fafF08 rescue is perfectly mirrored by
Hnt data with the added advantage of explicitly identifying dpphr4
heterozygous embryos (Fig. 5A-F, middle and right columns). The
data validates Lys738 as the ubiquitylation site associated with Faf
modulation of Med activity.
To firmly establish the conservation of Med deubiquitylation at
the level of the target lysine, we repeated the fafF08 rescue
experiment with two human Smad4 UASP transgenes (Smad4-wt
DEVELOPMENT
Fig. 4. faf zygotic and maternal mutant embryos display wild-type dpp and sog transcription and pMad activation. (A-F)In situ
hybridization to stage 5 embryos in lateral view. (A)Wild-type dpp is visible in the dorsal half of the embryo with slight ventral extensions in
terminal regions. (B)fafB4/fafBt5 maternal embryo from a female whose progeny survive beyond the faf-null phenotype (see Fig. 2B) displays wildtype dpp. (C)fafF08/fafBP zygotic embryo, from a cross generating cuticles with DV defects (see Fig. 1H), displays wild-type dpp. (D)Wild-type sog is
visible in the ventral half of the embryo but absent in terminal regions and the ventral-most 5-10%. (E)fafB4/fafB5 maternal mutant embryo displays
largely wild-type sog. (F)fafF08/fafBP zygotic mutant embryo displays wild-type sog. (G-I)Antibody labeling of stage 5 embryos shown in dorsal view.
(G)Wild-type pMad activation and localization is visible in a narrow stripe atop the dorsal-most region of the embryo with slightly wider expression
at the termini. (H)fafB4/fafB5 maternal mutant embryo displays normal pMad activation and a slightly broader pMad stripe. (I)fafF08/fafBP zygotic
mutant embryo exhibits wild-type pMad activation and localization.
Faf deubiquitylation in Dpp signaling
RESEARCH ARTICLE 2727
Fig. 5. Med-K738R rescues fafF08- and Med8
enhancement of dpphr4 better than Med-wt. (AF)Cuticles (left). Stage 10/11 embryos (middle) showing
endogenous Hnt (red), transgenic lacZ from the balancer
opposite dpphr4 (green, A-D; blue, E,F) and transgenic
eGFP (green, E,F). Red channel only (Hnt, right). Absence
of lacZ indicates the dpphr4 chromosome. (A)Wild-type
cuticle and embryo inheriting balancer chromosomes
from crosses between wild-type females and dpphr4
heterozygous males. (B)Ventralized cuticle and embryo
with significant reduction in Hnt generated by fafF08
dominant maternal enhancement of dpphr4.
(C)Ventralized cuticle and embryo with little Hnt indicate
that basal maternal expression of a Med-wt transgene
does not effectively rescue fafF08 enhancement of
dpphr4. (D)Wild-type cuticle and normal Hnt indicate
that basal maternal expression of a Med-K738R
transgene rescues fafF08 enhancement of dpphr4 better
than Med-wt. (E)Wild-type cuticle and normal Hnt
indicate that nos.Gal4 maternal expression of Med-wt
effectively rescues fafF08 enhancement of dpphr4.
(F)Wild-type cuticle and normal Hnt indicate that
nos.Gal4 maternal expression of Med-K738R also
effectively rescues fafF08 enhancement of dpphr4. (G)Bar
graph depicting transgenic rescue of fafF08 dominant
maternal enhancement of dpphr4 by Med-wt and MedK738R with basal and nos.Gal4 expression. The percent
of expected dpphr4 progeny obtained in crosses to wildtype and fafF08 heterozygous females (first and second
bars) is compared with matings of fafF08 heterozygous
females bearing four different transgene/promoter
combinations. Letters in each bar indicate data
corresponding to the panels above. There is 2.65-fold
better rescue with basal expression of Med-K738R
versus Med-wt (column D versus C). Numerical data are
in supplementary material Table S4A. (H)Transgenic
rescue of Med8 dominant maternal enhancement of
dpphr4 by Med-wt and Med-K738R with basal and
nos.Gal4 expression. There is 2.25-fold better rescue
with basal expression of Med-K738R versus Med-wt.
Numerical data are in supplementary material Table S4C.
fold better rescue with Smad4-K519R) was only slightly lower
than for Med-wt versus Med-K738R at basal levels (supplementary
material Fig. S7; Table S4D, Table S5D).
Overall, the rescue data indicate that Lys738 is the key residue by
which Med is regulated by the Faf deubiquitylase. These results
strongly suggest that regulation of Med/Smad4 deubiquitylation by
Faf/USP9X is a conserved molecular and developmental
mechanism regulating TGF responsiveness.
DISCUSSION
Faf deubiquitylase regulates Dpp signaling and
embryonic DV patterning
The existence of a ‘Smad4 ubiquitylation cycle’ has been recently
proposed in vertebrate model systems that requires the ubiquitin
ligase Ecto and the deubiquitylase USP9X. These studies suggested
that Ecto can monoubiquitylate Smad4 at Lys519 and that this
interferes with binding to R-Smads because Lys519 falls within a
crucial interaction surface. Subsequently, USP9X deubiquitylation
of Smad4 at Lys519 restores Smad4 function. The function of Ecto
as an inhibitor of TGF signaling was validated by mouse
knockout studies demonstrating that Ecto restrains early
Nodal/Smad4 signaling (Dupont et al., 2012). However, USP9X
activity has not yet been validated through any genetic test.
DEVELOPMENT
and Smad4-K519R). The relative performance of the two Smad4
transgenes (2.14-fold better rescue with Smad4-K519R) was nearly
identical to that of Med-wt versus Med-K738R at basal levels
(supplementary material Fig. S5; Table S4B, Table S5B).
A rescue assay with the Med8 null allele then controlled for the
possibility that Med-K738R is neomorphic. If Med acquired an
unusual activity via the K738R substitution that allowed it to rescue
fafF08 enhancement but this was not due to hyperactivity of normal
functions, then Med-K738R should not rescue a Med loss-offunction allele. If K738R leads to hyperactivity owing to ubiquitin
resistance, then it should rescue dpphr4 embryos from Med8
enhancement better than Med-wt.
Again, the most telling result was with the basal promoter (Fig.
5H; supplementary material Table S4C). Here Med-wt expression
increased the survival of dpphr4 progeny 1.6-fold. By contrast,
Med-K738R expression increased the survival of dpphr4 progeny
3.6-fold. Thus, Med-K738R performed 2.25-fold better in the
rescue of dpphr4 with basal expression. Once the transgenes were
driven at ectopic levels with nos.Gal4, Med-wt again closed the
gap in performance. These survival results are strongly supported
by cuticle assays and Hnt data (supplementary material Fig. S6,
Table S5C). We repeated the experiment with the Smad4
transgenes. The relative performance of the two transgenes (1.75-
2728 RESEARCH ARTICLE
Development 139 (15)
Here, we provide genetic evidence that reversible ubiquitylation
of Med can limit Dpp responsiveness. In faf mutants, defective
deubiquitylation renders embryonic cells unable to respond
appropriately to Dpp and results in defective DV patterning. This
conclusion is supported by multiple observations. (1) faf mutants
act as dominant maternal enhancers of dpp mutations leading to
defective DV axis formation in a manner comparable with Mad and
Med mutants. In addition, by reducing the levels of Dpp signaling
in sog mutants, faf mutants can partially rescue DV defects caused
by loss of sog, as shown for dpp mutants. (2) faf maternal mutant
genotypes generate a gradient of DV defects similar to that seen in
a dpp alleleic series. (3) faf mutants interact with Med mutants in
a non-reciprocal manner, strongly suggesting that faf acts in the
Dpp pathway by modifying Med activity. (4) Mutation of MedK738 and human Smad4-K519 render Med and Smad4 more active
then their wild-type counterparts and thus less susceptible to faf
mutation – most likely as a result of resistance to the activity of a
ubiquitin ligase that operates unopposed in the absence of Faf. We
summarize these findings in a model depicting the role of Faf in
Dpp DV signaling in Fig. 6.
An intriguing feature of our genetic analyses is that females
heterozygous for fafBP, a complete deletion of the faf locus, do not
enhance dpphr4, but females heterozygous for fafF08 and females
homozygous for fafEP381 do. Our interpretation is that faf maternal
activity must be reduced below one-half dose in the presence of
one-half dose of dpp zygotic activity to generate maternal
enhancement. Only homozygosity for the fafEP381 insertion and
heterozygosity for the fafF08 missense mutation in the catalytic
histidine reduce faf activity to that extent. Thus, fafF08 fits the
criterion (phenotypic effect is more severe than a deletion) for a
dominant-negative allele for faf functions in Dpp DV signaling.
From this perspective, the requirement for faf is less stringent than
the requirement for Mad and Med. For Mad and Med, reduction to
one-half dose is sufficient to engender dominant maternal
enhancement. The less stringent requirement for faf is consistent
with the fact that maternally contributed Med mutations, but not faf
mutations, generate synthetic lethality in double heterozygous
mutant individuals.
A ubiquitylation cycle required for morphogen
interpretation
The importance of a conserved zygotic extracellular system that
regulates embryonic DV differentiation via the generation of a
robust Dpp/BMP morphogen gradient is well known (e.g. Piccolo
et al., 1996). An equally relevant intracellular system employing
ubiquitin is now being recognized that acts in parallel to control
Dpp signal transduction (Xia et al., 2010). Our data extend this
recognition by showing that maternal intracellular regulation of
Med activity via ubiquitylation and deubiquitylation is a
fundamental feature of Dpp DV signaling.
Our study using faf mutants showed that a maternally
programmed intracellular balance of Med regulative ubiquitylation
and deubiquitylation is required for the zygotic extracellular system
to operate and can even compensate for an excess of Dpp, as shown
by the partial rescue of DV defects in sog and faf double mutants.
Furthermore, faf mutations do not affect pMad activation or dorsal
localization (except as a consequence of interfering with Med
activity in a Dpp feedback loop). This suggests that the level of
available Med (non-ubiquitylated) is a key quantitative variable, in
parallel with pMad that cells employ to interpret the Dpp gradient.
These data closely mirror results obtained in mouse embryo
knockouts for the Smad4 ubiquitin ligase Ecto. In this case, the
absence of a Smad4-inhibitory mechanism, and thus unrestrained
Smad4 activity, caused cells to respond to levels of extracellular
Nodal/intracellular phospho-Smad2 that would normally be too small
to activate gene expression. Thus, a global picture emerges whereby
cells keep Smad4 constantly in check, and that this is essential for
their ability to sense quantitative differences in R-Smad activity.
However, one wonders why do cells need such a cycle, instead
of simply fine-tuning Smad4 expression levels? One interesting
possibility is that Smad4 monoubiquitylation, which is promoted
by TGF signals in mammalian cells, might serve to continuously
clear active Smad complexes from promoters. This hypothesis is
supported by recent findings indicating that Ecto is recruited to
TGF target promoters in a Smad4-dependent fashion and that the
enzymatic activity of Ecto on Smad4 is promoted when Ecto is
bound to chromatin (Agricola et al., 2011). In this respect, Smad4
DEVELOPMENT
Fig. 6. Model for fat facets activity in Dpp DV signal transduction. Events downstream of a Dpp ligand in a dorsal cell from a blastoderm
stage embryo are depicted. Arrows represent the movement of information via phosphorylation (P) or changes in protein subcellular localization.
Cells contain pools of maternally contributed Mad (yellow), monoubiquitylated Med (purple with green ubiquitin attached) and Faf (blue). (A)Wild
type. Information flows from Dpp to transmembrane receptors to Mad via phosphorylation on serine (blue arrow). pMad then forms an activated
Smad complex with deubiquitylated Med in the nucleus where they drive transcription of rho. This results in normal DV axis formation. (B)fat facets
mutant. Information flows normally from Dpp to receptors to pMad but in the absence of Faf there is no deubiquitylated Med to form activated
Smad complexes. This prevents the activation of rho and leads to ventralization of the embryo.
ubiquitylation favors a dynamic state for R-Smads, keeping them
exposed to fluctuations in extracellular concentrations of TGF
ligands (Schmierer and Hill, 2007).
The Smad4 ubiquitin cycling model implies the existence of a
ubiquitin ligase for Med. One candidate is Bonus, the Drosophila
protein most closely related to the three vertebrate Tif1 proteins
(Beckstead et al., 2001). A second is Highwire, a ligase for Med at
the neuromuscular junction (McCabe et al., 2004). dSmurf, a ligase
shown to affect Mad but not Med in Dpp DV signaling, is not a
candidate (Liang et al., 2003). We tested bonus and highwire
mutants for DV phenotypes and found they are inconsistent with
those predicted for a Med ubiquitin ligase. Additional candidates
are currently under investigation.
In summary, our study reveals that Med deubiquitylation by Faf
is a conserved mechanism required for proper interpretation of the
Dpp morphogen gradient and embryonic DV axis formation.
Acknowledgements
We thank the following for valuable discussions, reagents and sharing or
pushing flies: Bloomington Stock Center, Iowa Hybridoma Bank, Estela
Arciniega, Kevin Cook, Eddy DeRobertis, Chip Ferguson, Janice Fischer, Mike
O’Connor, Nancy Tran and Robert Wisotzkey.
Funding
E.E. is supported by a Cassa di Risparmio di Padova e Rovigo (CARIPARO)
Foundation fellowship. The work was also supported by grants from the Italian
Association for Cancer Research to S.P. and S.D., from Comitato Promotore
Telethon to S.P., and from the Inter-Tribal Council of Arizona, The European
Network of Excellence (ENFIN) and Arizona State University to S.J.N.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.077206/-/DC1
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DEVELOPMENT
Faf deubiquitylation in Dpp signaling